External perturbations as well as population variability often result in broad fluctuations of growth and production rates of microorganisms. Close monitoring of a bioprocess is required to maximize process efficiency. Because of a lack of reliable on-line monitoring techniques, most often on-line bioprocess monitoring is limited to biogas analysis for oxygen and carbon dioxide content, while such key process parameters as substrate and product concentrations are only measurable off-line. Consequently, the results are available with a significant delay from the time of sampling. This delay leads to untimely process diagnosis as well as limits process control to pre-programmed feed strategies.
Recently developed on-line monitoring methods use flow injection analysis (FIA) techniques as well as near- and mid-infrared spectrometry (Tosi et al, Biotechnol. Prog., 19, 1816-1821 (2003)). While these techniques have been used successfully at the laboratory scale, high equipment cost is prohibitive for most industrial applications.
The use of fluorometry for rapid detection of fermentation imbalances and metabolic activities has already been demonstrated. Most often, fluorescence is measured by illuminating the sample at one wavelength and measuring fluorescence at another (higher) wavelength, i.e. a single excitation—single emission technique is used. In particular, NADPH-dependent fluorometry has been used for monitoring fermentation as well as aerobic and anaerobic wastewater treatment processes. However, bioreactor broth contains large amounts of proteins, amino acids, and other fluorescent compounds that interfere with NADPH-related fluorescence thus limiting industrial applications of single excitation—single emission fluorometry. The quality of monitoring can be improved by using multiple-excitation multiple-emission fluorescence measurements (e.g. Tartakovsky, B.; Lishman, L. A.; Legge, R. L., Water Research, 30 (12), 2941-2948 (1996)). In this technique, both excitation and emission wavelengths are varied to obtain two-dimensional spectra For this reason, this technique of fluorometric measurement, also employed herein, is often called two-dimensional fluorometry. The spectra are often processed using multivariate statistical analysis methods, such as Partial Least Square (PLS) regression, which provides a linear relationship between analytical measurements and multi-wavelength spectra.
To select a desired excitation wavelength, the light should pass through a monochromator or a filter wheel. The fluorescence signal (emission spectrum) can be measured by using a second monochromator or a filter wheel followed by a spectrometer. Alternatively, a close caption detector (CCD) array spectrometer can be used. Notably, the use of a monochromator or a filter wheel increases the setup cost and dimensions as well as it increases the scan time.
Light emitting diodes (LEDs) produce high intensity light in a narrow range (20-30 nm) of wavelengths. Thus, LEDs can be used for sample illumination at a fraction of the cost of conventional light sources equipped with monochromators or filter wheels. Indeed, some LED light sources are commercially available (LS-450, Ocean Optics Inc., Dunedin, Fla., USA). While LEDs are often used for illumination in the visible range of wavelengths, the use of LEDs for UV applications is relatively new. The UV LEDs are constantly improving with some newer LEDs emitting light at 350 nm (RLT350-30, ROITHNER, LASERTECHNIK, Vienna, Austria).
Fluorometers or similar devices have been described in patent literature as well. Some of the devices use LEDs. U.S. Pat. No. 6,825,927 (Goldman et al.) and U.S. Pat. No. 6,873,417 (Bahatt et al.) are examples of the prior art in this respect.
It is also known to use various optical waveguide arrangements to transfer light from the light source to the sample to be illuminated and to transfer the light (also fluorescence light) emitted by the sample to the measuring instruments. Exemplary patents are U.S. Pat. Nos. 6,791,687 and 6,166,804.
While the use of LEDs has reduced the cost of the fluorometric apparatus, there is still a room for improvement of the accuracy and reliability of the on-line monitoring of bioprocesses, e.g. food processing or wastewater treatment.